The present invention relates to a novel carboxypeptidase gene and the polypeptide encoded thereby. In particular, the present invention relates to the use of the present carboxypeptidase in the manufacture of cocoa flavor and/or chocolate.
It is known that in processing cacao beans the generation of the typical cocoa flavor requires two steps: a fermentation step, which includes air-drying of the fermented material, and a roasting step.
During fermentation, two major activities may be observed. First, the pulp surrounding the beans is degraded by micro-organisms with the sugars contained in the pulp being largely transformed to acids, especially acetic acid (Quesnel et al., J. Sci. Food. Agric. 16 (1965), 441-447; Ostovar and Keeney, J. Food. Sci. 39 (1973), 611-617). The acids then slowly diffuse into the beans and eventually cause an acidification of the cellular material. Second, fermentation also results in a release of peptides exhibiting differing sizes and a generation of a high level of hydrophobic free amino acids. This latter finding led to the hypothesis that proteolysis occurring during the fermentation step is not due to a random protein hydrolysis, but seems to be rather based on the activity of specific endoproteinases (Kirchhoff et al., Food Chem 31 (1989), 295-311). This specific mixture of peptides and hydrophobic amino acids is deemed to represent cocoa-specific flavor precursors.
Until now several proteolytic enzyme activities have been investigated in cacao beans and studied for their putative role in the generation of cocoa flavor precursors during fermentation.
An aspartic endoproteinase activity, which is optimal at a very low pH (pH 3.5) and inhibited by pepstatin A, has been identified. A polypeptide described to have this activity has been isolated and is described to consist of two peptides (29 and 13 kDa) which are deemed to be derived by self-digestion from a 42 kDa pro-peptide (Voigt et al., J. Plant Physiol. 145 (1995), 299-307). The enzyme cleaves protein substrates between hydrophobic amino acid residues to produce oligopeptides with hydrophobic amino acid residues at the ends (Voigt et al., Food Chem. 49 (1994), 173-180). The enzyme accumulates with the vicilin-class (7S) globulin during bean ripening. Its activity remains constant during the first days of germination and does not decrease before the onset of globulin degradation (Voigt et al., J. Plant Physiol. 145 (1995), 299-307).
Also, a cysteine endoproteinase activity had been isolated which is optimal at a pH of about 5. This enzymatic activity is believed not to split native storage proteins in ungerminated seeds. Cysteine endoproteinase activity increases during the germination process when degradation of globular storage protein occurs. To date, no significant role for this enzyme in the generation of cocoa flavor has been reported (Biehl et al., Cocoa Research Conference, Salvador, Bahia, Brasil, 17-23 Nov. 1996).
Moreover, a carboxypeptidase activity has been identified which is inhibited by PMSF, and thus belongs to the class of serine proteases. It is stable over a broad pH range with a maximum activity at pH 5.8. This enzyme does not degrade native proteins, but preferentially splits hydrophobic amino acids from the carboxy-terminus of peptides (Bytofet at., Food Chem. 54 (1995), 15-21).
During the second step of cocoa flavor production, the roasting step, the oligopeptides and amino acids generated at the stage of fermentation are obviously subjected to a Maillard reaction with reducing sugars present in fermented beans, eventually yielding substances responsible for the cocoa flavor as such.
In the art, there have been many attempts to artificially produce cocoa flavor.
Cocoa-specific aroma has been obtained in experiments wherein acetone dry powder (AcDP) prepared from unfermented ripe cacao beans was subjected to autolysis at a pH of 5.2 followed by roasting in the presence of reducing sugars. It was conceived that under these conditions preferentially free hydrophobic amino acids and hydrophilic peptides should be generated and the peptide pattern thus obtained was found to be similar to that of extracts from fermented cacao beans. An analysis of free amino acids revealed that Leu, Ala, Phe and Val were the predominant amino acids liberated in fermented beans or autolysis (Voigt et al., Food Chem. 49 (1994), 173-180). In contrast to these findings, no cocoa-specific aroma could) be detected when AcDP was subjected to autolysis at a pH of as low as 3.5 (optimum pH for the aspartic endoproteinase). Only few free amino acids were found to be released, but a large number of hydrophobic peptides were formed. When peptides obtained after the autolysis of AcDP at a pH of 3.5 were treated with carboxypeptidase A from porcine pancreas at pH 7.5, hydrophobic amino acids were preferentially released. The pattern of free amino acids and peptides was similar to that found in fermented cacao beans and to the proteolysis products obtained by autolysis of AcDP at pH 5.2. After roasting of the amino acids and peptides mixture as above, a cocoa aroma could be generated.
It has also been shown that a synthetic mixture of free amino acids alone, with a similar composition to that of the spectrum found in fermented beans, was incapable of generating cocoa aroma after roasting, indicating that both the peptides and the amino acids are important for this purpose (Voigt et al., Food Chem. 49 (1994), 173-180.
In view of the above data, a hypothetical model for the generation, during fermentation, of the said mixture of peptides and amino acids, i.e. the cocoa flavor precursors, had been devised (FIG. 1), where in a first step peptides having a hydrophobic amino acid at their end, are formed from storage proteins, which peptides are then further degraded to smaller peptides and free amino acids. To produce the said peptides having C-terminal hydrophobic amino acids, an aspartic endoproteinase activity related to that mentioned above seems to be involved. Yet, for splitting off hydrophobic amino acids from peptides formed in the preceding step the only known enzymatic activity, which might be considered in this respect, is that of a carboxypeptidase. However, such enzyme has not been isolated and studied in detail in cacao, and it is therefore still questionable which cacao enzyme might be responsible for the generation of hydrophobic amino acids required for cocoa flavor.
Though some aspects of cocoa flavor production have been elucidated, so far there is still a need in the art to fully understand the processes involved, so that the manufacture of cocoa flavor may eventually be optimized.